The goal of this project is to explore a new systems-level mechanism by which cells respond to stresses and exposures by regulating translation. Cells respond to xenobiotic exposures by linking external stimuli to changes in cell phenotype through signal transduction, transcriptional regulation, and protein 2 modifications. Using a unique computational and analytical platform, we recently discovered a new mechanism of translational control of the cell stress response in yeast, in which toxicant-induced reprogramming of dozens of modified ribonucleosides in tRNA regulates protein levels by promoting the selective translation of codon-biased mRNAs representing families of stress-response genes. The goal of the proposed studies is to test the hypothesis that translational regulation of gene expression by stress-specific coordinated changes in the dozens of tRNA modifications plays a role in the response of cells and tissues to xenobiotic exposure in mammals. Abundant preliminary data in rat and mouse models, including tissue from the National Toxicology Program's DrugMatrix, supports this hypothesis, with strong evidence that xenobiotic- induced changes in tRNA modifications, as well as enzymes that modify tRNA, are essential regulators of stress responses in complex tissues. Here we will use human cell and rat exposure models to firmly place tRNA reprogramming as a translational regulator of the response to xenobiotics. Specifically, we hypothesize that exposure to xenobiotics promotes toxicant-specific changes in tRNA modification patterns, and that the reprogrammed tRNAs regulate protein levels by way of selective translation of codon-biased stress response transcripts. We will test these hypotheses in two aims. Aim 1 focuses on in vitro analysis of tRNA reprogramming and proteomic changes in human lung and liver cells exposed to a battery of alkylating and oxidizing agents that overlap with our published yeast exposure results. This provides a systematic comparison of yeast and mammalian cell responses. Aim 2 moves the in vitro studies of Aim 1 to the in vivo setting to test the hypothesis that the translational respons mechanism occurs in complex tissues. We first build on preliminary studies of arsenite exposure in rats using tissues from the NTP DrugMatrix to define the link between tRNA reprogramming and codon-biased translation with iTRAQ proteomics. We then test the translational control model in rats exposed to inhaled formaldehyde in collaboration with Drs. Melanie Doyle- Eisele and Ben Moeller (Lovelace Resp. Res. Inst.) and Jim Swenberg (UNC). We previously showed that pulmonary exposure to formaldehyde generates protein and DNA adducts in nasal epithelium but not more distant lung and liver tissues, so we anticipate seeing formaldehyde-induced tRNA reprogramming and codon- biased translation in nasal tissue but not lung or liver, with nasal tissue matching the in vitro studies of Aim 1. These studies will provide critical insights into a novel mechanism of cell response to xenobiotic stress with direct relevance to human exposures. Future studies will translate this model to human drug exposures and inflammatory diseases.